Pharmacodynamic characterization of gemcitabine cytotoxicity in an in vitro cell culture bioreactor system
Department of Experimental and Clinical Pharmacology, College of Pharmacy and Comprehensive Cancer Center, University of Minnesota, 308 Harvard St SE, Minneapolis, MN 55455, USA. Cancer Chemotherapy and Pharmacology
(Impact Factor: 2.77).
03/2008; 61(2):291-9. DOI: 10.1007/s00280-007-0474-z
Gemcitabine, a pyrimidine nucleoside, is approved for the treatment of non-small cell lung cancer, pancreatic carcinoma, and breast cancer. Chemotherapy regimens are determined experimentally with static tissue culture systems, animal models, and in Phase I clinical trials. The aim of this study was to assess for gemcitabine-induced cell death following infusion of drug under clinically-relevant conditions of infusion rate and drug exposure in an in vitro bioreactor system.
To estimate an appropriate harvest time for cells from the bioreactor after drug treatment, we estimated the temporal relationship between gemcitabine treatment for 1 h and cell death at a later time point with monolayer growth assays (i.e., static culture). Afterward, 5.3 mg gemcitabine was infused over 0.5 h in the bioreactor, followed by mono-exponential decay, simulating patient concentration-time profiles (n = 4). Controls were run with drug-free media (n = 4). Cells were harvested from the bioreactor at a later time point and assessed for cell death by flow cytometry.
According to monolayer growth assay results, cytotoxicity became more apparent with increasing time. The E Max for cells 48 h after treatment was 50% and after 144 h, 93% (P = 0.022; t test), while flow cytometry showed complete DNA degradation by 120 h. Gemcitabine was infused in the bioreactor. The gemcitabine area under the concentration-time curve (AUC) was 56.4 microM h and the maximum concentration was 87.5 +/- 2.65 microM. Flow cytometry results were as follows: the G1 fraction decreased from 65.1 +/- 4.91 to 28.6 +/- 12% (P = 0.005) and subG1 increased from 14.1 +/- 5.28 to 42.6 +/- 9.78% (P = 0.004) relative to control. An increase in apoptotic cells was observed by TUNEL assay.
The in vitro bioreactor system will be expanded to test additional cell lines, and will serve as a useful model system for assessing the role of drug pharmacokinetics in delivery of optimized anticancer treatment.
Available from: Ayman Noreddin
- "In vitro models that incorporate these pharmacokinetic and pharmacodynamic (PK/PD) principles to optimize therapeutic response may be considered the method of choice for optimizing dosing schedules before translating data from static assays to animals and clinical trials  . The hollow fiber bioreactor was recently used to evaluate pharmacokinetic/pharmacodynamic (PK/PD) effects of gemcitibine in lung and breast cancers and to model HIV treatments    "
Readings in Advanced Pharmacokinetics - Theory, Methods and Applications, 04/2012; , ISBN: 978-953-51-0533-6
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ABSTRACT: Five-year survival for non-small cell lung cancer is 15%. Gemcitabine is a nucleoside analogue that inhibits ribonucleotide reductase and interferes with DNA replication. In this study, we sought to compare short versus continuous infusion gemcitabine in an in vitro bioreactor system using pharmacokinetic-guided dosing. Gemcitabine was infused over either 0.5 or 2.5h to produce concentration-time profiles that mimic those measured in biological samples (i.e., patient plasma). The effects of gemcitabine on the growth and survival of H2009 cells were examined using trypan blue staining, cell cycle analysis, TUNEL assay, and clonogenic assay. Data were analyzed with two ways analysis of variance. Maximum gemcitabine (Cmax) concentrations during the short infusion were 51.2+/-10.4 microM and for the continuous, 14.8+/-2.93 microM. Steady-state concentrations during the continuous infusions were 14.9+/-2.90 microM. Gemcitabine treatment resulted in a decrease for G1 fraction relative to controls. G2/M, subG1 and TUNEL were higher following gemcitabine relative to controls. Survival was approximately 20-fold higher following the short infusion compared with the continuous infusion (p = 0.0085). In conclusion, gemcitabine infused by this novel method induced apoptosis after both the short and continuous infusions, and long-term survival was significantly diminished following continuous compared with the short infusion.
Lung Cancer 12/2007; 58(2):196-204. DOI:10.1016/j.lungcan.2007.06.005 · 3.96 Impact Factor
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ABSTRACT: The use of in vitro screening tests for characterizing the activity of anticancer agents is a standard practice in oncology research and development. In these studies, human A2780 ovarian carcinoma cells cultured in plates are exposed to different concentrations of the compounds for different periods of time. Their anticancer activity is then quantified in terms of EC(50) comparing the number of metabolically active cells present in the treated and the control arms at specified time points. The major concern of this methodology is the observed dependency of the EC(50) on the experimental design in terms of duration of exposure. This dependency could affect the efficacy ranking of the compounds, causing possible biases especially in the screening phase, when compound selection is the primary purpose of the in vitro analysis. To overcome this problem, the applicability of a modeling approach to these in vitro studies was evaluated.
The model, consisting of a system of ordinary differential equations, represents the growth of tumor cells using a few identifiable and biologically relevant parameters related to cell proliferation dynamics and drug action. In particular, the potency of the compounds can be measured by a unique and drug-specific parameter that is essentially independent of drug concentration and exposure time. Parameter values were estimated using weighted nonlinear least squares.
The model was able to adequately describe the growth of tumor cells at different experimental conditions. The approach was validated both on commercial drugs and discovery candidate compounds. In addition, from this model the relationship between EC(50) and the exposure time was derived in an analytic form.
The proposed approach provides a new tool for predicting and/or simulating cell responses to different treatments with useful indications for optimizing in vitro experimental designs. The estimated potency parameter values obtained from different compounds can be used for an immediate ranking of anticancer activity.
Cancer Chemotherapy and Pharmacology 08/2008; 63(5):827-36. DOI:10.1007/s00280-008-0798-3 · 2.77 Impact Factor
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